Ischemic Wound Healing Research Articles

Tissue response to poly(ether)urethane-polydimethylsiloxane-fibrin composite scaffolds for controlled delivery of pro-angiogenic growth factors

The development of a scaffold able to mimic the mechanical properties of elastic tissues and to induce local angiogenesis by controlled release of angiogenic growth factors could be applied in the treatment of several ischemic diseases. For this purpose a composite scaffold made of a poly(ether)urethane–polydimethylsiloxane (PEtU–PDMS) semi-interpenetrating polymeric network (semi-IPN) and fibrin loaded growth factors (GFs), such as VEGF and bFGF, was manufactured using spray, phase-inversion technique. To evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, three different scaffold formulations were developed as follows: 1) bare PEtU–PDMS; 2) PEtU–PDMS/Fibrin; and 3) PEtU–PDMS/Fibrin + GFs. Scaffolds were characterized in vitro respect to their morphology, VEGF and bFGF release kinetics and bioactivity. The induction of in vivo angiogenesis after subcutaneous and ischemic hind limb scaffold implantation in adult Wistar rats was evaluated at 7 and 14 days by immunohistological analysis (IHA), while Laser Doppler Perfusion Imaging (LDPI) was performed in the hind limbs at 0, 3, 7, 10 and 14 days. IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU–PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU–PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU–PDMS/Fibrin + GFs scaffolds was higher than that of PEtU–PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU–PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU–PDMS scaffolds. LDPI showed that at 10 and 14 days the ischemic/non-ischemic blood perfusion ratio was significantly greater in the PEtU–PDMS/Fibrin + GFs than in the other scaffolds. In conclusion, this study showed that the semi-IPN composite scaffold acting as a pro-angiogenic GFs delivery system has therapeutic potential for the local treatment of ischemic tissue and wound healing.

Effects of isoniazid and niacin on experimental wound-healing

There is a need for effective treatments of ischemic wounds. Our aim was to test the hypothesis that systemic administration of isoniazid or niacin can enhance wound healing in ischemic as well as nonischemic tissues. One 8-mm, full-thickness wound was made in a standardized, ischemic skin flap and 1 in adjacent nonischemic skin on the back of male Sprague-Dawley rats. Starting just after wounding, twice-daily intraperitoneal isoniazid (10 mg/kg b.i.d.), xanthinol nicotinate (30 mg/kg), or saline (control) were given for 14 days. Wound-healing was monitored by planimetry and oxygen tension in periphery of the wound using a microcatheter probe. Cellular proliferation in granulation tissue was assessed by immunohistochemical detection of proliferating cell nuclear antigen. The angiogenic activity of isoniazid and niacin was assessed using in vitro and ex vivo models. Although wound ischemia was evident by decreased oxygen tension (26 +/- 10 mmHg; n = 9) compared with the adjacent nonischemic wounds (51 +/- 8 mmHg; n = 8), neither compound significantly influenced intracutaneous oxygen tension. Isoniazid (P < .0001), but not niacin, promoted ischemic wound-healing even though both compounds increased proliferation measured on day 14 (P < .01). In normal wounds, the cumulative change in relative wound area over 14 days was increased by niacin (P = .002), but not by isoniazid, although both niacin (P = .011) and isoniazid (P = .036) increased cellular proliferation. Neither isoniazid nor niacin showed activity in either an endothelial tube formation assay or organotypic angiogenic assay under normoxic conditions. Isoniazid was capable of stimulating wound-healing in ischemic tissue to the level of nonischemic wounds and might offer a novel treatment option for wounds associated with arterial insufficiency. Although active in normal wounds, niacin did not promote ischemic wound-healing.

Ultrasound accelerates healing of normal wounds but not of ischemic ones

To examine the influence of therapeutic ultrasound (US) on repair of standard and ischemic cutaneous lesions, full-thickness excisional wounds were made in rats and treated with a US 3 MHz, 0.5 W/cm(2) pulsed duty cycle. We used five experimental groups: control (received US powered off on the day of surgery, and on the second and fourth day), control US (received US on the day of surgery, and on the second and fourth day), ischemic (received US powered off on the day of surgery, and on the second and fourth day), ischemic US 3X (received US on the day of surgery, and on the second and fourth day) and ischemic US 5X (received US in the day of surgery, first, second, third and fourth day). The control US group showed acceleration in wound contraction 7 days after wounding, an increase in collagen density, and only focal inflammatory areas. Neo-epidermis formation was more advanced in the control US group than in the control one. Wound contraction was delayed in the ischemic group when compared with the control group as well as the ischemic US 3X group, was but slightly accelerated in the ischemic US 5X group when compared with the ischemic group 7 days after wounding. Reepithelialization was delayed in both ischemic US groups when compared with the ischemic group. The number of inflammatory cells was higher in both US ischemic groups. We conclude that US therapy accelerates wound healing in normal wounds and delays wound healing in ischemic wounds.

Simultaneous Characterization of Oxygen Transport into and through Porcine Skin Exposed to Oxygen-Saturated Water

Oxygen delivery to the skin is a promising approach for treatment of dermatological diseases (e.g. ischemic wound healing). However, characterization of oxygen transport into and through the skin exposed to oxygen carrier formulations has not been reported. In the present study, we developed an original lab-made static diffusion cell mounted with porcine skin enabling the assessment of oxygen uptake into the skin (i.e., oxygen penetration) and passage through the skin (i.e., oxygen permeation). Oxygen penetration and permeation were recorded by using an optical probe implanted into the skin tissue and a Clark-type electrode plunged into the receptor solution of the diffusion cells. Permeability parameters (i.e., maximal and steady-state flux; permeability coefficient) of oxygen were determined after a 2-hour application of oxygen-saturated water to either the skin surface (exogenous delivery) or the dermis (endogenous delivery). Similar experiments were performed by using intact or stripped skin in order to appreciate the role of the stratum corneum as oxygen barrier. Exogenous delivery of oxygen to skin tissue was found more effective than endogenous delivery through intact and stripped skin. However, exogenous oxygen permeation was found smaller than that determined from endogenous delivery. The upper layers of the skin would constitute a potential oxygen reservoir created by the high solubility of oxygen in epidermal lipids. Therefore, oxygen carrier formulations might significantly improve the oxygen status in the skin for further biological effects.

Quantifying tissue level ischemia: Hypoxia response element‐luciferase transfection in a rabbit ear model

Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE-luciferase-reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE-luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing.

In vivo properties of the proangiogenic peptide QK

The main regulator of neovascularization is Vascular Endothelial Growth Factor (VEGF). We recently demonstrated that QK, a de novo engineered VEGF mimicking peptide, shares in vitro the same biological properties of VEGF, inducing capillary formation and organization. On these grounds, the aim of this study is to evaluate in vivo the effects of this small peptide. Therefore, on Wistar Kyoto rats, we evaluated vasomotor responses to VEGF and QK in common carotid rings. Also, we assessed the effects of QK in three different models of angiogenesis: ischemic hindlimb, wound healing and Matrigel plugs. QK and VEGF present similar endothelium-dependent vasodilatation. Moreover, the ability of QK to induce neovascularization was confirmed us by digital angiographies, dyed beads dilution and histological analysis in the ischemic hindlimb as well as by histology in wounds and Matrigel plugs. Our findings show the proangiogenic properties of QK, suggesting that also in vivo this peptide resembles the full VEGF protein. These data open to new fields of investigation on the mechanisms of activation of VEGF receptors, offering clinical implications for treatment of pathophysiological conditions such as chronic ischemia.

Skin Perfusion Pressure is a Useful Tool for Evaluating Outcome of Ischemic Foot Ulcers with Conservative Therapy.

This study intended to confirm whether skin perfusion pressure (SPP) could predict the outcome of ischemic wound healing. Sixty-two limbs in 53 patients with conservative therapy were enrolled in this study. A SPP value of 40 mmHg was adopted as the criterion for making clinical decisions. The outcome one month after SPP measurement was classified as "improved" (diameter of ulcer decreased ≥ 20% or demarcation of gangrene became well defined) or "no change or worse" (others), and the fate of wound was classified as "healed" or "not healed". The evaluated influential factors on the outcome at one month included age, sex, presence of arteriosclerosis obliterans, collagen disease, hypertension, diabetes mellitus, hemodialysis, wound infection, wound management, and SPP ≥ 40 mmHg. Using a criterion of SPP ≥ 40 mmHg, the outcome at one month could be predicted with a sensitivity: of 75.0%, a specificity: of 82.6%, and an accuracy: of 80.6%. The receiver operating characteristic curve indicated our criterion to be appropriate. Logistic regression analysis showed SPP ≥ 40 mmHg to be an independent factor (P < 0.0001) with the odds ratio of 14.2 (95% CI 3.6-55.8). SPP, using a cutoff value of 40 mmHg, can predict the ischemic wound healing with conservative therapy.

Intradermal Delivery of Plasmid VEGF165 by Electroporation Promotes Wound Healing

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF(165) (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal injection followed by electroporation (EP) (pVEGFE+). Treatment with pVEGFE+ significantly increased VEGF expression for 5 days after delivery compared to injection of pVEGF without EP (pVEGFE-). The short-term increase in VEGF was sufficient to mediate an upregulation of endothelial nitric oxide synthase, an angiogenic factor that increases vascular permeability. pVEGFE+ significantly increased skin flap perfusion at both days 10 and 14 postoperatively. The observed increase in perfusion with pVEGFE+ correlated with an increase in skin flap healing and survival. Our results demonstrate that pVEGFE+ is a potential nonviral noninvasive therapy to increase perfusion and healing of skin flaps and ischemic wounds.

Surgical result of critical limb ischemia due to tibial arterial occlusion in patients with systemic scleroderma

To analyze the outcome after surgery for critical limb ischemia (CLI) due to tibial artery occlusion in patients with systemic scleroderma. The medical records of scleroderma patients with CLI due to tibial artery occlusion were reviewed with respect to demographic data and perioperative variables. Eight patients were identified at The University of Tokyo Hospital from 1991 to 2007. The underlying collagen disease was progressive systemic scleroderma in 6 patients and CREST syndrome in 2 patients. The subjects were 1 man and 7 women with a mean age of 68 years. While hypercoagulability including positive anticardiolipin antibodies was found in only 1 patient, all patients were antinuclear antibody (ANA) positive and 6 of 8 patients had a high titer of centromere-type ANA. Five underwent pedal artery bypass and 1 underwent distal peroneal artery bypass, while 2 underwent primary limb amputation. Although 1 patient with bypass had early graft occlusion (with subsequent below-knee amputation), the other 5 patients with patent grafts quickly achieved pain relief and initial wound healing. However, four of the five patent grafts developed graft occlusion several months after surgery, with severe intimal thickening at the anastomosis. As a result, 2 of 6 patients with bypass (totally 4 of 8 patients) underwent limb loss and 1 patient developed persistent recurrent ulcer. Bypass surgery in patients with scleroderma and CLI can be successful in achieving early pain relief and ischemic wound healing. However, the long-term effectiveness is limited with high rates of graft failure and limb loss.

Skin Perfusion Pressure is a Useful Tool for Evaluating Outcome of Ischemic Foot Ulcers with Conservative Therapy

Objective: This study intended to confirm whether skin perfusion pressure (SPP) could predict the outcome of ischemic wound healing.Patients and methods: Sixty-two limbs in 53 patients with conservative therapy were enrolled in this study. A SPP value of 40 mmHg was adopted as the criterion for making clinical decisions. The outcome one month after SPP measurement was classified as “improved” (diameter of ulcer decreased ≥ 20% or demarcation of gangrene became well defined) or “no change or worse” (others), and the fate of wound was classified as “healed” or “not healed”. The evaluated influential factors on the outcome at one month included age, sex, presence of arteriosclerosis obliterans, collagen disease, hypertension, diabetes mellitus, hemodialysis, wound infection, wound management, and SPP ≥ 40 mmHg.Results: Using a criterion of SPP ≥ 40 mmHg, the outcome at one month could be predicted with a sensitivity: of 75.0%, a specificity: of 82.6%, and an accuracy: of 80.6%. The receiver operating characteristic curve indicated our criterion to be appropriate. Logistic regression analysis showed SPP ≥ 40 mmHg to be an independent factor (P < 0.0001) with the odds ratio of 14.2 (95% CI 3.6–55.8).Conclusions: SPP, using a cutoff value of 40 mmHg, can predict the ischemic wound healing with conservative therapy.

Hyperbaric Oxygen Attenuates Apoptosis and Decreases Inflammation in an Ischemic Wound Model

The molecular mechanisms whereby hyperbaric oxygen (HBO) improves ischemic wound healing remain elusive. In this study, a rat model of wound ischemia was used to test the hypothesis that HBO enhances wound healing by modulating hypoxia-inducible factor-1alpha (HIF-1alpha) signaling. Male Sprague-Dawley rats underwent creation of a previously validated ischemic flap. Three groups underwent daily treatment: HBO (90 minutes, 2.4 atm); systemic administration of the free radical scavenger, N-acetylcysteine (NAC 150 mg kg(-1) intraperitoneal); control (neither HBO nor NAC). HBO treatment improved healing of the ischemic wounds. Analysis of ischemic wound tissue extracts demonstrated significantly reduced expression of HIF-1alpha, p53, and BNip3. Additionally, HBO increased expression of Bcl-2 while decreasing cleaved caspase-3. DNA fragmentation was abolished and the number of TUNEL-positive cells was reduced compared to the other groups. Vascular endothelial growth factor, cyclooxygenase-2, and neutrophil infiltration were reduced in ischemic wounds treated with HBO. These results indicate that HBO improves ischemic wound healing by downregulation of HIF-1alpha and subsequent target gene expression with attenuation of cell apoptosis and reduction of inflammation.

102 Inhibition of Reactive Oxygen Species Impairs Healing in a Model of Tissue Ischemia

Reactive oxygen species (ROS) play a major role in recruiting and activating inflammatory cells, inducing angiogenesis, fibroblast proliferation, and collagen synthesis, all processes essential to wound healing. However, the same mediators have been implicated in pulmonary and hepatic fibrosis, vascular occlusion, and necrotic tissue damage. We have previously shown that hyperbaric oxygen caused a prolonged elevation of VEGF and delayed wound healing in a rat model of tissue ischemia. In this study we examine the effect of the ROS scavenger, n-acetylcysteine (NAC), on ischemic wound healing. NAC is a virtually nontoxic free radical scavenger that has been used in many experimental studies to block ROS-mediated signaling. Methods: Male Sprague-Dawley rats underwent creation of the ischemic flap previously validated by this laboratory. This model provides two ischemic and two nonischemic excisional wounds. Rats were treated daily with HBO for 90 minutes at 2.4 atm, HBO plus 150 mg/kg NAC intraperitoneal, or control (neither HBO nor NAC). Wounds were analyzed for surface area, lactate, and VEGF. Results: The HBO/NAC-treated animals had markedly impaired healing of their ischemic wounds at day 7 (0.105 ± .005 cm vs. 0.068 ± .002 cm for ctl and 0.064 ± .006 for HBO) and a twofold elevation of VEGF (120 pg/mg protein vs. 60 pg/mg protein). Lactate levels were not altered by NAC treatment and non-ischemic wounds showed no difference in healing, lactate, or VEGF. Conclusion: Reactive oxygen species are required for wound healing. Our initial hypothesis was that the delay in wound closure with HBO treatment was due to excess ROS and that NAC would improve healing. The finding of elevated VEGF and delayed wound healing in the presence of a potent free radical scavenger suggests that blocking ROS signaling prevents the normal mitogenic response to VEGF. Additional studies to examine the VEGF receptor and tyrosine kinase activation will further elucidate the mechanism of ROS signaling in response to HBO treatment.

023 HRE-Luciferase Transfection Quantifies Tissue Level Ischemia in a Rabbit Ear Wound Model

The Hypoxia Inducible Factor (HIF) system has been characterized as the principal tissue level response to hypoxia. Posttranslational regulation of HIF-1alpha has been reported to act though a Hypoxia Responsive Element (HRE) promoter region on a range of hypoxia-induced genes. A plasmid was constructed consisting of a fivefold HRE repeat conjugated to a luciferase gene, used as a marker for HRE activation. Plasmid HRE-luciferase was then transfected into a well-established ischemic rabbit ear wound healing model. Ischemia was induced using a variety of published models for interruption of arterial inflow and compared with a nonischemic control. Central artery, caudal artery and rostrocaudal artery models were tested for induction of ischemia. Tissue specimens were harvested from the transfected areas and solubilized. Luminescence of each specimen was measured using a standard luminometer to quantify luciferase induction. To provide correlation on a regional level, tissue level oxygen tension was measured directly for each wound model. Profound and statistically significant differences were found in the induced luciferase production. Marked differences in the tissue hypoxia between the various ischemic wound healing models correlated in graded fashion with the ischemia induced on an anatomic basis. The least ischemic model showed no significant difference in hypoxia readings versus control. The intermediate model showed a significant fourfold greater ischemia signaling. The most ischemic model showed a highly significant 72-fold greater ischemic response compared to controls. The use of gene transfection is described as a sensitive and effective method for quantification of tissue hypoxia at a cellular level in ischemic wounds. Acknowledgments: This study was funded by the Wound Healing Research Laboratory.

027 Exogenous Administration of SMAD3 by an Adenoviral Vector Significantly Affects Ischemic Tissue Repair

Introduction: Chronic, nonhealing wounds are often observed in tissues with poor oxygen supply. Impaired reepithelialization is a hallmark of these wounds; however, the pathogenesis of the retarded reepithelialization in chronic, ischemic wounds remains poorly understood. Transforming Growth Factor beta (TGF-beta) is involved in both normal and hypoxic wound healing response and exogenous overexpression of Smad3, a TGF-beta signaling intermediate, has been known to accelerate reepithelialization. In a recent study, Ad-Smad3 injection in the rabbit ear dermal ulcer model showed enhanced reepithelialization and granulation tissue area suggesting a positive effect of Smad3 on wound healing. However, little is known about the role of Smad3 in the ischemic wound healing process. In this study we examined the effect of Smad3 in an ischemic wound model. Methods: Ad-Smad3 or LacZ (108 pfu/wound) empty vector was injected in either ear of White New Zealand Rabbits. Twenty-four hours later, these ears were rendered ischemic using an established model and four 7-mm full-thickness punch wounds were made on each ear. Results: Histological evaluation showed a highly significant increase in reepithelialization, epithelial ingrowth and percentage of epithelialization in Ad-Smad3 transfected wounds versus ischemic wounds transfected with LacZ-empty vector (p < 0.01). Conclusion: Our data confirm the enhancing effect of Smad3 on reepithelialization in an ischemic wound model. The deficiency in reepithelialization, as evident in chronic ischemic wounds, could thus be ameliorated by excess Smad3. Therapeutic interventions using overexpressed Smad3 may improve wound healing through accelerated epithelialization in chronic wounds.

Predictive factors of nipple areolar complex invasion in breast cancer patients with mastectomy

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF165 (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal injection followed by electroporation (EP) (pVEGFE+). Treatment with pVEGFE+ significantly increased VEGF expression for 5 days after delivery compared to injection of pVEGF without EP (pVEGFE−). The short-term increase in VEGF was sufficient to mediate an upregulation of endothelial nitric oxide synthase, an angiogenic factor that increases vascular permeability. pVEGFE+ significantly increased skin flap perfusion at both days 10 and 14 postoperatively. The observed increase in perfusion with pVEGFE+ correlated with an increase in skin flap healing and survival. Our results demonstrate that pVEGFE+ is a potential nonviral noninvasive therapy to increase perfusion and healing of skin flaps and ischemic wounds.

ビーグル犬における高気圧酸素療法の骨延長術での治癒促進効果について

Distraction osteogenesis has been widely used even in the craniofacial region. A long fixation time during the consolidation period, however, is a major clinical disadvantage. Hyperbaric oxygen (HBO) has been used to improve healing in ischemic wounds. We have recently started applying hyperbaric oxygen to cleft palate patients after maxillary distraction, but there is little basic evidence. We hypothesized that hyperbaric oxygen would enhance the healing of distraction osteogenesis in the cleft palate model in dogs. A bony segment including a canine was transported proximally into an artificial bone defect in the left palate. Three dogs were treated with hyperbaric oxygen for 20 days just after the distraction and three other dogs underwent only the distraction process (control group). Blood flow of the canine pulp in the bone segment was monitored using a laser Doppler flowmeter throughout the experiment. All the dogs were sacrificed on day 100, and radiological analysis using peripheral quantitative CT and histomorphometric evaluations were performed. Blood flow in the HBO-treated group recovered to the original level about 30 days faster than in the control group (p<0.05). Cortical bone mineral density was significantly higher at the distraction site in the HBO-treated group than in the control group (p<0.05). The histomorphometric analysis revealed that the newly formed bone area was also larger in the HBO-treated group than in the control group (p<0.05). These results suggest that hyperbaric oxygen treatment could be useful for early removal of the distraction device in distraction osteogenesis.

Persistent ischemia impairs myofibroblast development in wound granulation tissue: A new model of delayed wound healing

We describe a new animal model designed to assess the impact of ischemia on wound healing. Eight patterns of arterial lesion in the limb were first tested in 24 Wistar rats. Resection of the external iliac artery down to the femoral artery at the level of the knee was chosen as the reference model and performed on the left limb of 45 rats; the right limb was used as the control. Skin wounds measuring 1.2 x 0.8 cm were created on both feet. Ischemia was assessed by blood flow measurement, which decreases dramatically in the ischemic limb. A significant delay in wound closure with a decrease in wound contraction was observed in the ischemic limb. Myofibroblast quantification showed a significant delay in appearance as well as a decrease in the number of these cells in the ischemic wound. Vascular endothelial growth factor-A appearance and evolution were qualitatively similar in both situations. However, collagen type I mRNA was markedly decreased in ischemic granulation tissue 10 days after wounding. These findings suggest that decreased wound contraction plays an important role in delayed ischemic wound healing, probably due to reduced myofibroblast development and activity.

Angiogenesis and vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of bone marrow–derived progenitor cell mobilization and homing

During embryonic development, the vasculature is among the first organs to form and is in charge of maintaining metabolic homeostasis by supplying oxygen and nutrients and removing waste products. As one would expect, blood vessels are critical not only for organ growth in the embryo but also for repair of wounded tissue in the adult. An imbalance in angiogenesis (a time-honored term that globally refers to the growth of new blood vessels) contributes to the pathogenesis of numerous malignant, inflammatory, ischemic, infectious, immune, and wound-healing disorders. This review focuses on the central role of the growth of new blood vessels in ischemic and diabetic wound healing and defines the most current nomenclature that describes the neovascularization process in wounds. There are now two well-defined, distinct, yet interrelated processes for the formation of postnatal new blood vessels, angiogenesis, and vasculogenesis. Reviewed are recent new data on vasculogenesis that promise to advance the field of wound healing.

Alteration of Smad3 signaling in ischemic rabbit dermal ulcer wounds

Impaired reepithelialization is a hallmark of chronic, ischemic wounds; however, the pathogenesis of the delayed reepithelialization in these wounds remains poorly understood. Transforming growth factor beta is involved in both the normal and hypoxic wound-healing response and exogenous overexpression of Smad3, which has been known to accelerate reepithelialization. Recently, it was shown in the rabbit ear dermal ulcer model that Ad-Smad3 injection enhanced reepithelialization and granulation tissue formation suggesting a positive effect of Smad3 on wound healing. However, little is known about the role of Smad3 in the ischemic wound healing process. In this study, we examined the effect of Smad3 in an ischemic wound model. Ad-Smad3 or Ad-LacZ (10(8) pfu/wound) was injected into either ear of white New Zealand rabbits. Twenty-four hours later, these ears were rendered ischemic using an established model, and four 7 mm full-thickness punch wounds were made on each ear. Histological evaluation showed a significant increase in reepithelialization parameters in Ad-Smad3-transfected wounds (p<0.01). In contrast, granulation tissue parameters were not affected by Smad3 in ischemia. Smad4 and Smad7 mRNA-expression was not affected by Smad3 overexpression. Connective tissue growth factor protein was up-regulated under ischemic conditions but was unaffected by Smad3 transfection in both ischemic and nonischemic wounds. Our results suggest an enhancing effect of Smad3 on reepithelialization in an ischemic wound model that, in turn, might provide novel therapeutic options. Furthermore, the lack of alteration of Smad-dependent intermediates by Smad3 overexpression suggests the activation of Smad-independent pathways in ischemia.

Effects of Topical Glutathione Treatment in Rat Ischemic Wound Model

Oxidative stress secondary to ischemia can cause physiopathologic changes that adversely affect wound healing. In this experimental study, we hypothesized that the topical use of esterified glutathione, a well-known antioxidant, can minimize the effects of oxidative stress by an increase in intracellular glutathione and accelerate wound healing by increasing the contraction capacity of fibroblasts and preventing keratinocytes from apoptosis in a rat ischemic wound model. Experimental models were divided into 3 groups as treatment, control, and healthy. Bipedicled flaps were elevated from the dorsum of the rats, and 6-mm punch wounds were created at the end of the first day when the ischemia is most apparent. Wounds were followed histopathologically and immunohistochemically, and matrix metalloproteinase (MMP)-1 and tissue inhibitors of metalloproteinase (TIMP-1) levels were measured by ELISA. Samples were collected at 0, 5, 8, 10, and 12 days. Histopathologic evaluation revealed significant extracellular matrix deposition and reepithelization every fifth day in treatment and healthy groups when compared with control group. Immunohistochemical evaluation revealed increased apoptosis in basal keratinocytes in the control group when compared with the other groups. The evaluation of the samples collected at 5 and 8 days revealed increased MMP-1 levels in treatment and control groups, but the increase in TIMP-1 levels was more significant than MMP-1 levels in treatment group. MMP-1/TIMP-1 ratio was significantly low in the treatment group.Our results showed that topical GSH treatment can reduce oxidative stress, and the reestablishment of the MMP-1/TIMP-1 ratio gives way to adequate and regular extracellular matrix production and reepithelization. It is concluded that esterified GSH, which is experimentally shown to be effective in ischemic wound healing, can be used clinically in ischemic wounds.